The ATP-dependent carboxylate-amine/thiol ligase superfamily is known to contain enzymes catalyzing the formation of various types of peptide, such as D-alanyl-D-alanine, polyglutamate, and ␥-peptide, but, curiously, no enzyme synthesizing ␣-dipeptides of L-amino acids is known. We attempted to find such an enzyme. By in silico screening based on the consensus sequence of the superfamily followed by an in vitro assay with purified enzyme to avoid the degradation of the peptide(s) synthesized, ywfE of Bacillus subtilis was found to code for the activity forming L-alanyl-L-glutamine from L-alanine and L-glutamine with hydrolysis of ATP to ADP. No AMP was formed, supporting the idea that the enzyme belongs to the superfamily. Surprisingly, the enzyme accepted a wide variety of L-amino acids. Among 231 combinations of L-amino acids tested, reaction products were obtained for 111 combinations and 44 kinds of ␣-dipeptides were confirmed by high-performance liquid chromatography analyses, while no tripeptide or longer peptide was detected and the D-amino acids were inert. From these results, we propose that ywfE encodes a new member of the superfamily, L-amino acid ligase.

A large-scale production system of uridine 5'-diphospho-galactose (UDP-Gal) has been established by the combination of recombinant Escherichia coli and Corynebacterium ammoniagenes. Recombinant E. coli that overexpress the UDP-Gal biosynthetic genes galT, galK, and galU were generated. C. ammoniagenes contribute the production of uridine triphosphate (UTP), a substrate for UDP-Gal biosynthesis, from orotic acid, an inexpensive precursor of UTP. UDP-Gal accumulated to 72 mM (44 g/L) after a 21 h reaction starting with orotic acid and galactose. When E. coli cells that expressed the alpha1,4-galactosyltransferase gene of Neisseria gonorrhoeae were coupled with this UDP-Gal production system, 372 mM (188 g/L) globotriose (Galalpha1-4Galbeta1-4Glc), a trisaccharide portion of verotoxin receptor, was produced after a 36 h reaction starting with orotic acid, galactose, and lactose. No oligosaccharide by-products were observed in the reaction mixture. The production of globotriose was several times higher than that of UDP-Gal. The strategy of producing sugar nucleotides by combining metabolically engineered recombinant E. coli with a nucleoside 5'-triphosphate producing microorganism, and the concept of producing oligosaccharides by coupling sugar nucleotide production systems with glycosyltransferases, can be applied to the manufacture of other sugar nucleotides and oligosaccharides.

L-Homophenylalanine (L-Hph) is a useful chiral building block for synthesis of several drugs, including angiotensin-converting enzyme inhibitors and the novel proteasome inhibitor carfilzomib. While the chemoenzymatic route of synthesis is fully developed, we investigated microbial production of L-Hph to explore the possibility of a more efficient and sustainable approach to L-Hph production. We hypothesized that L-Hph is synthesized from L-Phe via a mechanism homologous to 3-methyl-2-oxobutanoic acid conversion to 4-methyl-2-oxopentanoic acid during leucine biosynthesis. Based on bioinformatics analysis, we found three putative homophenylalanine biosynthesis genes, hphA (Npun_F2464), hphB (Npun_F2457), and hphCD (Npun_F2458), in the cyanobacterium Nostoc punctiforme PCC73102, located around the gene cluster responsible for anabaenopeptin biosynthesis. We constructed Escherichia coli strains harboring hphABCD-expressing plasmids and achieved the fermentative production of L-Hph from L-Phe. To our knowledge, this is the first identification of the genes responsible for homophenylalanine synthesis in any organism. Furthermore, to improve the low conversion efficiency of the initial strain, we optimized the expression of hphA, hphB, and hphCD, which increased the yield to ϳ630 mg/liter. The L-Hph biosynthesis and L-Leu biosynthesis genes from E. coli were also compared. This analysis revealed that HphB has comparatively relaxed substrate specificity and can perform the function of LeuB, but HphA and HphCD show tight substrate specificity and cannot complement the LeuA and LeuC/LeuD functions, and vice versa. Finally, the range of substrate tolerance of the L-Hph-producing strain was examined, which showed that m-fluorophenylalanine, o-fluorophenylalanine, and L-tyrosine were accepted as substrates and that the corresponding homoamino acids were generated.

In spite of its clinical and nutritional importance, L-alanyl-L-glutamine (Ala-Gln) has not been widely used due to the absence of an efficient manufacturing method. Here, we present a novel method for the fermentative production of Ala-Gln using an Escherichia coli strain expressing L-amino acid ␣-ligase (Lal), which catalyzes the formation of dipeptides by combining two amino acids in an ATP-dependent manner. Two metabolic manipulations were necessary for the production of Ala-Gln: reduction of dipeptide-degrading activity by combinatorial disruption of the dpp and pep genes and enhancement of the supply of substrate amino acids by deregulation of glutamine biosynthesis and overexpression of heterologous L-alanine dehydrogenase (Ald). Since expression of Lal was found to hamper cell growth, it was controlled using a stationary-phase-specific promoter. The final strain constructed was designated JKYPQ3 (pepA pepB pepD pepN dpp glnE glnB putA) containing pPE167 (lal and ald expressed under the control of the uspA promoter) or pPE177 (lal and ald expressed under the control of the rpoH promoter). Either strain produced more than 100 mM Ala-Gln extracellularly, in fed-batch cultivation on glucose-ammonium salt medium, without added alanine and glutamine. Because of the characteristics of Lal, no longer peptides (such as tripeptides) or dipeptides containing D-amino acids were formed.Glutamine (Gln) is the most abundant free-form amino acid in human cells. Gln is not an essential amino acid for humans, but it is considered to be a conditionally essential amino acid because of its central role in nitrogen metabolism (7,40). In spite of such nutritional importance, Gln has hardly been used as a component of parenteral nutrition due to its low solubility and instability in solution. One approach to overcome the physicochemical limitations of Gln is to supply the amino acid as a dipeptide by conjugation with other amino acids. Alanylglutamine (Ala-Gln) is the most suitable Gln-containing dipeptide. Since there have been a number of reports on the safety and effectiveness of this dipeptide (2, 7), the clinical and nutritional importance of Ala-Gln has already been established.Several methods for producing Ala-Gln have been described; chemical or enzymatic condensation of protected alanine (Ala) and Gln (3,22,24,43) and a chemical synthetic process via D-2-chloropropionyl-glutamine (32). These methods, however, have not been satisfactory in regard to costeffectiveness and quality. Most methods need protection steps and sometimes deprotection steps, lowering production efficiency (8). The protecting reactions also trigger racemization of the substrate amino acid(s), which results in the formation of an undesired by-product, D-alanyl-glutamine. These chemical and enzymatic methods have also been suggested to form other by-products, such as different dipeptides (for example, alanyl-glutamic acid) or longer oligopeptides (for example, tripeptides) (32,43).Recently, we identified a new enzyme named L-amino acid ␣-ligase (Lal)...

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